Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers

ABSTRACT

This invention relates to the use of mixed mode chromatography for purification of a protein from a mixture containing other materials, including fragmented or aggregated antibodies, host cell proteins, DNA, endotoxin, and/or virus. This invention further relates to the integration of such a method into a multi-step procedure with other fractionation methods for purification of antibodies or other proteins suitable for in vivo applications.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/970,296, filed Jan. 7, 2009 now U.S. Pat. No. 7,691,980,issued Apr. 6, 2010, which claims benefit of priority to each of U.S.Provisional Patent Application No. 60/879,484, filed Jan. 9, 2007; U.S.Provisional Patent Application No. 60/905,696, filed Mar. 8, 2007; andU.S. Provisional Patent Application No. 60/913,162, filed Apr. 20, 2007,each of which is incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods for enhancing purification of proteinsby mixed mode chromatography. In certain embodiments, the enhancementmay permit the chromatography method to bind greater amounts of proteinor virus, thereby improving productivity and facilitating its practicaluse for initial capture of protein from unpurified preparations. Inother embodiments, the enhancement may permit more effective separationof protein from other material (including, but not limited to, otherproteins, DNA, endotoxin, or virus) and other contaminants. In otherembodiments, the enhancement may permit more effective separation ofnon-aggregated antibody from aggregated antibody.

BACKGROUND OF THE INVENTION

Mixed mode chromatography involves the use of solid phasechromatographic supports that employ multiple chemical mechanisms toadsorb proteins or other solutes. Examples include but are not limitedto chromatographic supports that exploit combinations of two or more ofthe following mechanisms: anion exchange, cation exchange, hydrophobicinteraction, hydrophilic interaction, hydrogen bonding, pi-pi bonding,and metal affinity.

Mixed mode chromatography supports provide unique selectivities thatcannot be reproduced by single mode chromatography methods such as ionexchange, however method development is complicated, unpredictable, andmay require extensive resources. Even then, development of usefulprocedures may require long periods of time, as exemplified byhydroxyapatite.

Hydroxyapatite is a crystalline mineral of calcium phosphate with astructural formula of Ca₁₀(PO₄)₆(OH)₂. Chemically reactive sites includepairs of positively charged calcium atoms and triplets of negativelycharged phosphate groups. The interactions between hydroxyapatite andproteins are multi-modal, hence its classification as a mixed modesupport. One mode of interaction involves metal affinity of proteincarboxyl clusters for crystal calcium atoms. Another mode of interactioninvolves cation exchange of positively charged protein amino residueswith negatively charged crystal phosphates (Gorbunoff, AnalyticalBiochemistry 136 425 (1984); Kawasaki, J., Chromatography 152 361(1985)).

The individual contributions of the two mechanisms to the binding andelution of a particular protein can be controlled in part by the choiceof salts used for elution. The cation exchange interaction can becontrolled with a gradient of any salt, including phosphate salts,sulfates, nitrates, or chlorides, specifically including sodium chlorideand potassium chloride. The calcium affinity mode is inert to mostcommonly used non-phosphate salts. Thus proteins that bind byinteraction with the calcium groups on hydroxyapatite cannot be elutedby sodium chloride alone. They can be eluted with phosphate salts.

Hydroxyapatite is commonly used for purification of antibodies,especially from partially purified preparations. The column is usuallyequilibrated and the sample applied in a buffer that contains a lowconcentration of phosphate. Adsorbed antibodies are often eluted in anincreasing gradient of phosphate salts (Gagnon, Purification Tools forMonoclonal Antibodies, Chapter 5, Validated Biosystems, Tucson, ISBN0-9653515-9-9 (1996); Luellau et al., Chromatography 796-165 (1998)).Gradients of phosphate combined with non-phosphate salts such as sodiumchloride have also been used for protein purification, includingantibody purification (Freitag, “Purification of a recombinanttherapeutic antibody by hydroxyapatite chromatography,” Oralpresentation, 2d International Hydroxyapatite Conference, San Francisco(2001)). One such approach involves the application of a gradient ofsodium chloride or potassium chloride while a low level of phosphate isheld constant (Kawasaki et al., Eur. J. Biochem., 155-249 (1986); Sun,“Removal of high molecular weight aggregates from an antibodypreparation using ceramic hydroxyapatite,” Oral presentation, 3rdInternational Hydroxyapatite Conference, Lisbon (2003); Gagnon et al.,“Practical issues in the use of hydroxyapatite for industrialapplications,” Poster BIOT 322, 232nd meeting of the American ChemicalSociety, San Francisco, (2006)[http://www.validated.com/revalbio/pdffiles/ACS_CHT 0_(—)02.pdf]; Wyethet al., U.S. Patent Application, Publication No. WO/2005/044856 (2005)).This approach has also been applied to antibody purification withfluorapatite (Gagnon et al., “Simultaneous removal of aggregate, leachedprotein A, endotoxin, and DNA from protein A purified monoclonal IgGwith ceramic hydroxyapatite and ceramic fluorapatite,” OralPresentation, Wilbio Conference on Purification of Biological Products,Santa Monica, (2005)[http://www.validated.com/revalbio/pdffiles/PBP_(—)2005.pdf]).Fluorapatite is prepared by fluoridating hydroxyapatite. Thissubstitutes fluoride for the hydroxyl groups creating a mineral with thestructural formula Ca₁₀(PO₄)₆F₂.

Hydroxyapatite has been shown to yield a high degree of purification ina single step. However, the presence of phosphate and other ions mayreduce binding capacity to a degree that makes either hydroxyapatite orfluorapatite economically unsuitable as capture methods (Gagnon et al.,Hydroxyapatite as a Capture Method for Purification of MonoclonalAntibodies, IBC World Conference and Exposition, San Francisco (2006)[http://www.validated.com/revalbio/pdffiles/Gagnon_IBCSF06.pdf]). Thisprevents them from being competitive with capture methods that arerelatively unaffected by phosphate and salt concentration, such asprotein A affinity chromatography.

Most non-antibody protein contaminants elute before antibodies, butantibodies from different clones elute in different areas of the elutionprofile and may therefore overlap to varying degrees with contaminatingproteins. Known methods for enhancing the separation are oftenineffective and may be undesirable for economic reasons as well. Forexample, a shallow linear elution gradient can be applied but this hasthe negative side effects of increasing the buffer volume and processtime, and it may still fail to achieve the desired purity.

Hydroxyapatite has been shown to be effective for removal of degradedforms of antibodies such as fragments, but selectivity is highlydependent on whether elution is conducted with a chloride gradient orwith a phosphate gradient.

Hydroxyapatite and fluoroapatite have been shown to be effective forremoval of aggregates from many antibody preparations. Antibodyaggregates usually elute after antibodies but may coelute withantibodies to varying degrees. Aggregate removal is important becauseaggregates are known to contribute to nonspecific interactions thatreduce the shelf stability, sensitivity, accuracy, and reproducibilityof analytical results in conjunction with in vitro diagnosticapplications. Aggregates are known to mediate adverse pharmacologicaleffects, such as complement activation, anaphylaxis, or formation oftherapy-neutralizing antibodies in conjunction with in vivo therapeuticapplications. Aggregates also reduce purification efficiency byrequiring additional steps to achieve adequately low aggregate levels inthe final product. Elution of hydroxyapatite and fluorapatite withchloride gradients at low fixed concentrations of phosphate has beenshown to be more effective than simple phosphate gradients, but eventhis approach may not be sufficient for all antibody preparations.

Various other mixed mode chromatography methods for antibodypurification have been introduced in recent years. Examples ofcommercial products exploiting mixed mode functionalities include butare not limited to MEP Hypercel (Pall Corporation); Capto-MMC,Capto-Adhere, Capto-Q, Capto-S (GE Healthcare); and ABx (J.T. Baker).These products have varying degrees of ability to remove aggregates,host cell proteins, DNA, and virus from antibody preparations, but aswith hydroxyapatite, method development is complex and unpredictable,and their utility as capture methods is often limited by low capacity.

Aqueous-soluble nonionic organic polymers are known in the field ofprotein purification for their ability to precipitate proteins,including antibodies. They have also been reported to increase theretention of proteins in protein A affinity chromatography and ionexchange chromatography (Gagnon, Purification Tools for MonoclonalAntibodies, Chapter 5, Validated Biosystems, Tucson, ISBN 0-9653515-9-9(1996); Gagnon et al., “Multiple mechanisms for improving binding of IgGto protein A,” Poster, BioEast, Washington D.C., (1992); Gagnon et al.,“A method for obtaining unique selectivities in ion exchangechromatography by adding organic solvents to the mobile phase,” Posterand Oral presentation, 15th International Symposium on HPLC of Proteins,Peptides, and Polynucleotides, Boston (1995)[http://www.validated.com/revalbio/pdffles/p3p95iec.pdf]). Such organicpolymers include but are not limited to polyethylene glycol (PEG),polypropylene glycol, polyvinylpyrrolidone, dextran, cellulose, andstarch, of various polymer molecular weights. PEG is an organic polymerwith a structural formula of HO—(CH₂—CH₂—O)_(n)—H. In addition to itsapplications for protein fractionation, it is known as a proteinstabilizer appropriate for use in pharmaceutical formulations.

SUMMARY OF THE INVENTION

The present invention relates to methods of purifying proteins(including but not limited to antibodies) from a preparation containingproteins to be purified by contacting said preparation with a mixed modechromatography support in the presence of an aqueous-soluble (i.e.,hydrophilic) nonionic organic polymer. Applicant surprisingly found thatthe presence of a nonionic organic polymer enhances binding capacity forvirus or protein on mixed mode chromatography supports. In the case ofprotein capture this enables higher levels of productivity to beachieved, and expands the range of methods that may be considered fortheir initial capture from unpurified preparations. In the case ofvirus, this enables more effective viral clearance from the proteinproduct Applicant further surprisingly found that the presence ofnonionic organic polymer preferentially enhances the retention ofantibody on mixed mode chromatography supports in comparison to mostcontaminating proteins, thereby enabling novel selectivity for improvedremoval of non-antibody proteins. Applicant further surprisingly foundthat the presence of nonionic organic polymer preferentially enhancesretention of aggregated antibody and other very large molecules (e.g.,viruses) on mixed mode chromatography supports in comparison tonon-aggregated antibody, thereby enabling novel selectivity, superiorseparation, and superior removal capacity of large-contaminantsincluding but not limited to virus. Most surprisingly, applicant foundthat the effects of soluble nonionic organic polymer on antibody bindingand elution behavior are relatively uniform among different mixed modechromatography methods, despite dramatic differences in their respectivenative selectivities. This is particularly valuable because it permits auniform approach for applying the invention to any given antibodypreparation, regardless of the composition of a particular mixed modechromatography support.

The protein preparation may be applied to the mixed mode chromatographysupport in a variety of concentrations of nonionic organic polymer. Insome embodiments, the concentration of nonionic organic polymer rangesfrom about 0.01% to 50%. In some embodiments, the concentration ofnonionic organic polymer is between 0.1 to 50%, 1%-50%, 3%-50%, 5%-50%,1%-70%, 1%-10%, 0.1%-10%, etc.

The concentration of nonionic organic polymer may be held constant or itmay be altered throughout the course of the separation (including butnot limited to in a gradient of increasing or decreasing concentration,or with step-wise changes in concentration).

The average molecular weight of the nonionic organic polymer can vary.In some embodiments, the average molecular weight ranges from, about 100to 10,000 daltons, e.g., 100-1000, 1000-5000, etc.

In some embodiments, the nonionic organic polymer is PEG. In someembodiments, the PEG has an average molecular weight of 6,000 daltonsand/or is applied at a concentration of 0.01 to 7.5%. In someembodiments, the PEG has an average molecular weight of 2,000 daltonsand/or is applied at a concentration of 0.01 to 15%.

In some embodiments, a protein preparation is applied to the mixed modechromatography support in the presence of nonionic organic polymerthereby resulting in increased protein- (including but not limited to,antibody-) binding and/or virus-binding capacity of the support. Thus,in some embodiments, a sufficient concentration of nonionic organicpolymer is present in the relevant buffer to delay elution by 10% (or,e.g., 20%, 50%, etc.). For example, where an increasing gradient of asalt is used to elute the protein (e.g., antibodies), the center of theeluted antibody peak occurs at a concentration of the salt at least 10%higher than would occur in the absence of the nonionic organic polymer.

In some embodiments, a protein preparation is applied to the mixed modechromatography support under conditions that permit the binding of virusor non-aggregated protein and contaminants, with fractionation of thevirus or non-aggregated protein being achieved subsequently by changingthe conditions such that the non-aggregated protein (e.g., antibody) iseluted while contaminants remain bound to the support. This mode ofapplication is often referred to as “bind-elute” mode. In someembodiments, one or more proteins in the preparation is eluted andcollected while virus remains bound to the support, thereby purifyingthe protein and reducing or eliminating viral contamination of theprotein.

In some embodiments of bind-elute mode, the concentration of nonionicorganic polymer is held constant during elution, while the pH ismodified or the concentration of eluting salts is increased.

In some embodiments of bind-elute mode, the concentration of nonionicorganic polymer is increased during elution, while the pH is modified orthe concentration of eluting salts is increased.

In some embodiments of bind-elute mode, the concentration of nonionicorganic polymers may be decreased during elution, while the pH and saltconcentration are held constant.

The protein preparation may be applied to the mixed mode chromatographysupport under conditions that prevent the binding of non-aggregatedprotein while permitting the binding of aggregated protein and otherlarge-molecules (including, e.g., viruses). This mode of application isoften referred to as “flow-though” mode. Bound virus or aggregates oflarge molecules may be eluted subsequently upon treatment with anappropriate buffer.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations specified in the claims.

The invention may be practiced in combination with one or more othermethods, including but not limited to protein A and other forms ofaffinity chromatography, anion exchange chromatography, cation exchangechromatography, hydrophobic interaction chromatography, other mixed modechromatography, and non-chromatographic methods. It is within theability of a person of ordinary skill in the art to develop appropriateconditions for these methods and integrate them with the inventiondescribed herein to achieve purification of a particular antibody.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute partof this specification, and together with the description, serve toexplain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an application of the invention in which nonionicorganic polymer is used to increase dynamic binding capacity of anantibody preparation on hydroxyapatite.

FIG. 2 illustrates an application of the invention in which nonionicorganic polymer is used to improve removal of non-antibody protein(“NAP”), aggregated antibody, and other contaminants from an antibodypreparation by hydroxyapatite chromatography.

FIG. 3 illustrates an application of the invention in which nonionicorganic polymer is used to improve the separation of aggregated andnon-aggregated antibody from an antibody preparation by hydroxyapatitechromatography.

FIG. 4 illustrates an application of the invention in which nonionicorganic polymer is used to improve the separation of aggregated antibodyby hydroxyapatite chromatography. The arrows in the figure identifypeaks of aggregated antibody.

FIG. 5 illustrates separation of antibody aggregates from antibodymonomers. The left graph shows that while each of two differentantibodies (“chimera (a)” and “chimera (b)”) had different elutioncharacteristics, on hydroxyapatite, the addition of PEG affected them innearly the same way. This demonstrates that the effect of PEG dominatesthe selectivity of the system. This in turn illustrates that the methodis broadly applicable to different antibodies and results in separationof aggregates from monomers that does not occur in the absence of PEG.The right graph shows that the degree of separation of monomer andaggregate peaks is enhanced in the presence of even modest amounts ofPEG. R values of 1.5 and over generally indicates a baseline between thepeaks, meaning that one peak can be fully eluted before the second peakbegins elution.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Terms are defined so that the invention may be understood more readily.Additional definitions are set forth throughout the detaileddescription.

“Single mode support” refers to a chromatographic solid phase thatinvolves substantially a single chemical adsorption mechanism. Examplesinclude cation exchangers and anion exchangers.

“Mixed mode chromatography support” refers to a chromatographic solidphase that substantially involves a combination of two or more chemicalmechanisms. In some embodiments, the combination results in uniqueselectivities such that it is able to achieve fractionation amongantibodies, antibody aggregates, antibody fragments, other proteins,DNA, endotoxin, and virus, that cannot be achieved by a single modesupport. Examples of chemical mechanisms that can be combined in mixedmode supports include but are not limited to cation exchange, anionexchange, hydrophobic interaction, hydrophilic interaction, hydrogenbonding, pi-pi bonding, and metal affinity. The solid phase can be aporous particle, nonporous particle, membrane, or monolith.

“Nonionic organic polymer” refers to an aqueous-soluble uncharged linearor branched polymer of organic composition. Examples include, but arenot limited to dextran, starch, cellulose, polyvinylpyrrolidone,polypropylene glycol, and polyethylene glycol of various molecularweights. Polyethylene glycol has a structural formulaHO—(CH₂—CH₂—O)_(n)—H. Examples include, but are not limited tocompositions with an average polymer molecular weight ranging from 100to 10,000 daltons. The average molecular weight of commercial PEGpreparations is typically indicated by a hyphenated suffix. For example,PEG-6000 refers to a preparation with an average molecular weight ofabout 6,000 daltons.

“Antibody” refers to an immunoglobulin, composite, or fragmentary formthereof. The term may include but is not limited to polyclonal ormonoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM,derived from human or other mammalian cell lines, including natural orgenetically modified forms such as humanized, human, single-chain,chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitrogenerated antibodies. “Antibody” may also include composite formsincluding but not limited to fusion proteins containing animmunoglobulin moiety. “Antibody” may also include antibody fragmentssuch as Fab, F(ab′)2, Fv, scFv, Fd, dAb, Fc and other compositions,whether or not they retain antigen-binding function.

“Protein preparation” refers to any composition containing a protein tobe purified. In some cases, the protein to be purified is an antibody.“Antibody preparation” refers to any composition containing anon-aggregated antibody. Said preparation may also contain antibodyfragments and/or aggregates. Non-antibody proteins and othercontaminants, potentially including but not limited to nucleic acids,endotoxin, and virus may also be present.

“Aggregate” refers to an association of at least two antibodies andoften more (e.g., 5, 10, 20 or more antibodies). The association may beeither covalent or non-covalent without respect to the mechanism bywhich the antibodies are associated. The association may be directbetween the antibodies or indirect through other molecules that link theantibodies together. Examples of the latter include but are not limitedto disulfide linkages via other proteins, hydrophobic associations vialipids, charge associations via DNA, affinity associations via leachedprotein A, or mixed mode associations via multiple components.

“Complex” refers to an association of an antibody with one or morenon-antibody molecules. The association may be either covalent ornon-covalent without respect to the mechanism of association. Examplesinclude but are not limited to associations with other proteins, lipids,DNA, leached protein A, or multiple components.

“Bind-elute mode” as it relates to the invention herein, refers to anoperational approach to chromatography in which the buffer conditionsare established so that both a target protein (e.g., non-aggregatedantibody) and undesired contaminants bind to the mixed modechromatography support when the protein preparation is applied in thepresence of nonionic organic polymer. Fractionation of intactnon-aggregated protein is achieved subsequently by changing theconditions such that the target of interest is eluted from the supportwhile contaminants remain bound. These contaminants may optionally beremoved by an appropriate cleaning buffer.

“Flow-through mode” as it relates to the invention herein, refers to anoperational approach to chromatography in which the buffer conditionsare established so that intact non-aggregated protein to be purifiedflows through the mixed mode chromatography support upon application,while aggregates and other large molecules (including viruses) areselectively retained, thus achieving their removal.

“Preparative applications” refers to situations in which the inventionis practiced for the purpose of obtaining purified protein for research,diagnostic, therapeutic, or other applications. Such applications may bepracticed at any scale, ranging from milligrams to kilograms of antibodyper batch.

“In-line dilution” refers to a method of chromatographic sampleequilibration that can be used to avoid antibody precipitation before itcan bind to the column. Advance addition of nonionic organic polymer toan antibody preparation may cause antibody to precipitate over a periodof time. Such precipitates may cause problems if applied to a packedchromatography column. In-line dilution adds nonionic organic polymer tothe antibody preparation as it is being pumped onto the column.Conditions may be used under which pre-column contact time of antibody(or other target) to nonionic organic polymer is too brief for antibody(or other target) precipitation to occur.

B. Materials

1. Mixed Mode Chromatography Supports

Various mixed mode chromatography media are available commercially, anyof which can be used to practice of this invention. Commerciallyavailable examples include but are not limited to ceramic hydroxyapatite(CHT) or ceramic fluorapatite (CFT), MEP-Hypercel™, Capto-MMC™,Capto-Adhere™, Capto-S™, Capto-Q™, and ABx™.

“Hydroxyapatite” refers to a mixed mode support comprising an insolublehydroxylated mineral of calcium phosphate with the structural formulaCa₁₀(PO₄)₆(OH)₂. Its dominant modes of interaction are phosphoryl cationexchange and calcium metal affinity.

“Fluorapatite” refers to a mixed mode support comprising an insolublefluoridated mineral of calcium phosphate with the structural formulaCa₁₀(PO₄)₆F₂. Its dominant modes of interaction are phosphoryl cationexchange and calcium metal affinity.

“Ceramic” hydroxyapatite (CHT) or “ceramic” fluorapatite (CFT) refer toforms of the respective minerals in which nanocrystals are agglomeratedinto particles and fused at high temperature to create stable ceramicmicrospheres suitable for chromatography applications. Commercialexamples of ceramic hydroxyapatite include, but are not limited to CHTType I and CHT Type II. Commercial examples of fluorapatite include, butare not limited to CFT Type I and CFT Type II. Unless specified, CHT andCFT refer to roughly spherical particles of any average diameter,including but not limited to about 10, 20, 40, and 80 microns. Thechoice of hydroxyapatite or fluorapatite, the type, and average particlediameter can be determined by the skilled artisan.

In some embodiments, the mixed-mode chromatography support exploits acombination of anion exchange and hydrophobic interactionfunctionalities. Examples of such supports include, but are not limitedto, MEP-Hypercel™.

In some embodiments, the mixed-mode chromatography support exploits acombination of cation exchange and hydrophilic interactionfunctionalities. Examples of such supports include, but are not limitedto, Capto-S™.

In some embodiments, the mixed-mode chromatography support exploits acombination anion exchange and hydrophilic interaction functionalities.Examples of such supports include, but are not limited to, Capto-Q™.

In some embodiments, the mixed-mode chromatography support exploits acombination of cation exchange, anion exchange, and hydrophobicinteraction functionalities. Examples of such supports include, but arenot limited to, ABx™.

In some embodiments, the mixed-mode chromatography support exploits acombination of anion exchange and hydrophobic interactionfunctionalities with potential for hydrogen bonding and pi-pi bonding.Examples of such supports include, but are not limited to,Capto-Adhere™.

In some embodiments, the mixed-mode chromatography support exploits acombination of cation exchange and hydrophobic interactionfunctionalities with potential for hydrogen bonding and pi-pi bonding.Examples of such supports include, but are not limited to, Capto-MMC™.

The invention may be practiced in a packed bed column, afluidized/expanded bed column containing the hydroxyapatite orfluorapatite, and/or a batch operation where the mixed mode support ismixed with the antibody preparation for a certain time.

In some embodiments, a mixed mode chromatography support is packed in acolumn.

In some embodiments, the mixed mode support is packed in a column of atleast 5 mm internal diameter and a height of at least 25 mm. Suchembodiments are useful, e.g., for evaluating the effects of variousconditions on a particular antibody or other target protein.

Another embodiment employs the mixed mode support, packed in a column ofany dimension required to support preparative applications. Columndiameter may range from less than 1 cm to more than 1 meter, and columnheight may range from less than 1 cm to more than 30 cm depending on therequirements of a particular application.

It will be appreciated that the present invention is not limited to theabove heights and diameters. Appropriate column dimensions can bedetermined by the skilled artisan.

2. Proteins

Protein (antibody or non-antibody protein) preparations to which theinvention can be applied can include, but are not limited to, unpurifiedor partially purified proteins (including, e.g., antibodies) fromnatural, synthetic, or recombinant sources. Unpurified protein apreparations can come from various sources including, but not limitedto, plasma, serum, ascites, milk, plant extracts, bacterial lysates,yeast lysates, or conditioned cell culture media. Partially purifiedpreparations can come from unpurified preparations that have beenprocessed by at least one chromatography, precipitation, otherfractionation step, or any combination of the foregoing. Thechromatography step or steps can employ any method, including but notlimited to affinity, anion exchange, cation exchange, protein Aaffinity, hydrophobic interaction, immobilized metal affinity, ormixed-mode chromatography. The precipitation step or steps can includeany method including, but not limited to, salt or PEG precipitation.Other fractionation steps can include, but are not limited to,crystallization or membrane filtration. The proteins (including but notlimited to antibodies) can be PEGylated or alternatively, not PEGylated.

Exemplary non-antibody proteins include any protein with a therapeutic,industrial, diagnostic, or other effect. Such proteins can benaturally-occurring or recombinant. The proteins can be generated intissue or cell cultures or isolated from animals or plants.

Proteins (e.g., antibodies or non-antibody proteins) can be purified ineither flow-through mode or bind-elute mode, as described herein.

3. Nonionic Organic Polymers

Various commercially available nonionic organic polymers can be used topractice the invention. Examples include, but are not limited topolyethylene glycol (PEG), polypropylene glycol, cellulose, dextran,starch, and polyvinylpyrrolidone.

PEG provides a general model for behavior of soluble nonionic organicpolymers within the context of the invention. Thus, while PEG isdiscussed in the following text, one should recognize that theinformation applies equally to other nonionic polymers, including butnot limited to those specifically listed herein.

The invention may be practiced with PEG with an average polymermolecular weight ranging from about 100 to about 10,000 Daltons.Exemplary PEG includes PEG having an average molecular weight of, e.g.,200, 300, 400, 500, 900, 1000, 1400, 2000, 3300, 4500, 8000, 10000,14000, etc. In some embodiments, the PEG has an average weight between400-1000, 200-1000, 400-2000, or 1000-5000. A wide variety of differentPEGs are available from, e.g., Aldrich.

PEG or other organic polymers can be linear or branched polymers.

Lower molecular weight PEGs will generally require a higherconcentration to achieve an effect similar to higher molecular weightPEGs.

Lower concentrations of a given molecular weight of PEG are generallyused to enhance the binding of larger proteins (e.g., larger antibodiesand fusion proteins) as well as viruses compared to concentrations tothe concentration of PEG to result in the same amount of enhancedbinding of smaller proteins. For example, IgM, with an approximatemolecular weight of about 960 kD, will generally require a lowerconcentration of PEG to achieve a certain degree of binding enhancementthan IgG, with an approximate molecular weight of 160 kD. Retention ofaggregates, complexes, and other large molecule contaminants willgenerally be enhanced to a greater degree than the unaggregated forms ofthe proteins from which they are derived.

Lower concentrations of PEG will be generally required to enhance thebinding of molecules that are strongly retained by the mixed modechromatography support, compared to the concentration for PEG to achievethe same enhanced binding for molecules that are weakly retained.

The effects described in the two preceding paragraphs will generally becompound: the retention of large molecules that are strongly retained inthe absence of nonionic organic polymer will be enhanced more byapplication of the invention than molecules that are smaller and weaklyretained, smaller and strongly retained, or larger and weakly retained.

In some embodiments, PEG with an average molecular weight of about 6,000Daltons is employed in a concentration range from 0.0-7.5% to separateintact IgG from aggregated forms (see figures).

In some embodiments, PEG with an average molecular weight of about 2,000Daltons is employed in a concentration range from 0.0-15.0% to separateintact IgG from aggregated forms.

The identity, appropriate average molecular weight, and concentration ofthe organic polymer to practice the invention can be determined by theskilled artisan.

C. Description of the Method

In preparation for contacting the protein (e.g., antibodies) preparationwith the mixed mode support, in some embodiments, the chemicalenvironment inside the column is equilibrated. This is commonlyaccomplished by flowing an equilibration buffer through the column toestablish the appropriate pH; conductivity; identity, molecular weight,and concentration of nonionic organic polymer; and other pertinentvariables.

In some embodiments, the protein (e.g., antibody) preparation is alsoequilibrated to conditions compatible with the column equilibrationbuffer before the invention can be practiced. This generally consists ofadjusting the pH, salt concentration; the identity, average molecularweight and concentration of nonionic organic polymer.

In one embodiment, nonionic organic polymer is added to directly theprotein (e.g., antibody) preparation before it is applied to the column.However this potentially limits the amount of nonionic organic polymerthat can be used because an excessive concentration of polymer may causethe protein (e.g., antibody) or other components of the preparation toprecipitate before the sample can be loaded on the column.

In another embodiment, nonionic organic polymer is added to the protein(e.g., antibody) preparation by means of in-line dilution. This allows ahigher percentage of nonionic organic polymer to be employed, becausethe pre-column contact time of the polymer with the sample is reduced toseconds or less. Appropriate conditions can be determined by the skilledartisan.

In some embodiments, after the column and protein (e.g., antibody)preparation is equilibrated, the protein preparation may be contactedwith the column. The protein preparation may be applied at a linear flowvelocity in the range of, for example, about 50-300 cm/hr. Appropriateflow velocity can be determined by the skilled artisan.

In one embodiment of the flow-through mode, non-aggregated protein(e.g., antibody) flows through the column and is collected, while virusand/or aggregated protein binds to the column. Optionally, the proteinpreparation is followed with a wash buffer, usually of the samecomposition as the equilibration buffer. This displaces remainingnon-aggregated protein from the column so that it can be collected.Retained aggregates may optionally be removed from the column with anappropriate cleaning buffer.

Flow-through mode conditions can be developed depending on the specificprotein desired. Without intending to limit the scope of the invention,the following description is provided as a guide for developingflow-through conditions as desired for a particular protein. In someembodiments, a PEG (or other soluble nonionic organic polymer)concentration is identified whereby aggregates or other undesirablecomponents of the preparation would bind to the mixed-mode column andnon-aggregated protein would not. For example, phosphate, sodiumchloride, other salts, or a combination thereof, can be tested at avariety of concentrations and conditions, initially in the absence ofsoluble nonionic organic polymer to identify conditions at which targetand contaminant (e.g., antibody/antibody aggregate or protein/virus)elute. Increasing (or decreasing) amount of soluble nonionic organicpolymer is then injected into the sample until the appropriateconcentration is identified at which the target (e.g., non-aggregatedantibody, non-antibody protein, etc.) flows through but the aggregateand/or other contaminant remains bound to the column.

In one embodiment of an application conducted in bind-elute mode, bothaggregated and non-aggregated protein and/or virus bind to the column.In some embodiments, sample application is followed with a wash buffer,usually of the same composition as the equilibration buffer (optionallylacking, or having a reduced amount of, the soluble nonionic organicpolymer). This removes unretained contaminants from the column.Non-aggregated protein is then eluted from the column under conditionsthat leave aggregated protein or other contaminants such as virusesbound to the column. Retained contaminants may optionally be removedfrom the column with an appropriate cleaning buffer.

In one embodiment of the bind-elute mode, the wash buffer may have aformulation different than the equilibration buffer.

The skilled artisan will recognize that various strategies ofmanipulating nonionic organic polymer concentration during elution willpermit successful application of the invention.

In one embodiment of the bind-elute mode, the concentration of nonionicorganic polymer is held constant during elution, while the pH is alteredand/or the concentration of eluting salts is increased. For example, insome embodiments, the target (e.g., antibody or other protein) is elutedin, e.g., 0.1-0.8 M phosphate, or optionally, at relatively lowconcentrations of phosphate (e.g., less than 0.2 or less than 0.1 M) butwith additional salts (e.g., NaCl, KCl, etc.), e.g., at 0.3 M or more,0.5 M or more, etc.

In another embodiment of the bind-elute mode, the concentration ofnonionic organic polymer is decreased during elution, while the pH andconcentration of eluting salts is held constant.

In another embodiment of the bind-elute mode, the concentration ofnonionic organic polymer is increased during elution, while theconcentration of eluting salts is also increased. This embodiment willoften give the best separation between non-aggregated and aggregatedprotein and/or virus because the later-eluting aggregates/virusexperience a higher concentration of nonionic organic polymer,preferentially enhancing their retention and increasing their separationfrom non-aggregated protein.

After use, the mixed mode column may optionally be cleaned, sanitized,and stored in an appropriate agent, and optionally, re-used.

In some embodiments, the invention will have a beneficial effect onremoval of other contaminants, including but not limited to, nucleicacids, endotoxin, virus, and complexes of antibody with leached proteinA.

Exemplary viruses that can be removed by the methods of the inventioninclude, e.g., membrane-encapsulated viruses as well as non-encapsulatedviruses. Contaminating viruses may derive from the cell lines used toproduce a given protein product, or from exogenous contamination.Viruses that can be removed by the methods of the invention may alsoinclude viruses that are deliberately introduced as markers for thepurpose of quantifying and validating the ability of a givenpurification method to effectively remove the introduced virus.Exemplary viruses to be removed include but not limited to, HIV, HBV,HCV, and HPV, and non-human animal viruses, including but not limitedto, minute virus of mouse (MVM), and murine leukemia virus (MuLV).

D. Additional Optional Steps

The present invention may be combined with other purification methods toachieve higher levels of purification. Examples include, but are notlimited to, other methods commonly used for purification of proteins,such as affinity chromatography (e.g., such as protein A affinitychromatography for purification of antibodies), anion exchangechromatography, cation exchange chromatography, hydrophobic interactionchromatography, immobilized metal affinity chromatography, additionalmixed mode chromatography methods, precipitation, and filtration.

Removal of residual organic polymer from purified protein, if desired,can be accomplished without need for a specific nonionic organic polymerremoval step. In some embodiments, the nonionic organic polymer can beomitted from the wash and elution steps, so that the eluted antibody issubstantially polymer-free. Alternatively, if the target protein isbound to a chromatography medium in a subsequent process step, residualnonionic organic polymer will pass through the column. This approachwill work with most ion exchangers, mixed mode, and affinity methods.Removal of residual nonionic polymer can also be facilitated byemploying polymers of low average molecular weight so that they can beremoved by diafiltration or other buffer exchange methods.

EXAMPLES

It is well known in the art of antibody purification that considerablevariation in chromatographic behavior is encountered from one antibodypreparation to another. This includes variation in the composition andproportion of non-antibody proteins, antibody fragments, and aggregatesthat contaminate various antibody preparations, as well as variation inthe individual retention characteristics of different antibodies. Thismakes it necessary to customize the buffer conditions to apply theinvention to its best advantage in each situation. This may involveadjustment of pH, the concentration of salts, the concentration pHbuffering components, choice of the identity, average molecular weightand concentration of nonionic organic polymer. Appropriate levels forthe various parameters and components can be determined systematicallyby a variety of approaches. The following examples are offered forillustrative purposes only.

Example 1

Bind-elute mode, enhancement of binding capacity. See, FIG. 1. A columnof hydroxyapatite, CHT Type II, 20 micron, 5 mm diameter, 50 mm height,is equilibrated at a linear flow rate of 300 cm/hr with 5 mM sodiumphosphate at pH 6.7. A monoclonal antibody preparation IgG1, 1 mg/ml)previously purified by protein A affinity chromatography is equilibratedto the same conditions and applied to the column. The effluent ismonitored for UV absorbance at 280 nm to characterize the bindingcapacity of the column. The column is then cleaned with about 600 mMpotassium phosphate, pH 6.7. The run is repeated but with about 2.5%PEG-6000 added to the sample and column equilibration buffer. Differentmolecular weights or concentrations of PEG, and variations in otherparameters, may be evaluated in subsequent iterations to determine theformulation that provides the best results for the particular antibody.Other nonionic organic polymers may be evaluated as well.

Example 2

Bind-elute mode, enhanced removal of non-antibody protein contaminantsand aggregates from a preparation of unpurified monoclonal antibody. Acolumn of hydroxyapatite, CHT Type II, 20 micron, 5 mm diameter, 5 cmheight, is equilibrated at a linear flow rate of 300 cm/hr with 5 mMsodium phosphate at pH 6.7. An unpurified antibody preparation(unpurified chimeric IgG1) is applied to the column, washed withequilibration buffer, then eluted with a gradient to 5 mM sodiumphosphate, 2.0 M sodium chloride, pH 6.7. The run is repeated but elutedwith linear gradient to about 5 mM sodium phosphate, 2.0 M sodiumchloride, and about 5% PEG-6000, and cleaned with 500 mM sodiumphosphate at 300 cm/hr (1 ml/min) (see, FIG. 2). In a subsequentiteration, the run is repeated except that the concentration of PEG-6000in the gradient endpoint buffer is increased to 3.75%. Differentmolecular weights or concentrations of PEG, and variations in otherparameters, may be evaluated in subsequent iterations to determine theformulation that provides the best results for the particular antibody.Other nonionic organic polymers may be evaluated as well.

Example 3

Bind elute mode, enhanced removal antibody aggregates from a preparationor protein A purified monoclonal antibody. See, FIG. 3. A column ofhydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 5 cm height, isequilibrated at a linear flow rate of 300 cm/hr with 5 mM sodiumphosphate at pH 6.7. The partially purified antibody preparation (humanIgG1 purified by protein A affinity chromotography) is applied to thecolumn, washed with equilibration buffer, then eluted with a lineargradient to 5 mM sodium phosphate, 2.0 M sodium chloride, pH 6.7 at alinear flow rate of 600 cm/hr (2 ml/min). The run is repeated but elutedwith a gradient to about 5 mM sodium phosphate, 2.0 M sodium chloride,and up to 7.5% PEG-6000. Different molecular weights or concentrationsof PEG, and variations in other parameters, may be evaluated insubsequent iterations to determine the formulation that provides thebest results for the particular antibody. Other nonionic organicpolymers may be evaluated as well.

Example 4

Bind elute mode, enhanced removal antibody aggregates from a preparationor protein A purified monoclonal antibody. See, FIG. 4. A column ofhydroxyapatite, CHT Type I, 20 micron, 5 mm diameter, 5 cm height, isequilibrated at a linear flow rate of 300 cm/hr (1 ml/min) with 10 mMsodium phosphate at pH 7.0. The partially purified antibody preparation(chimeric IgG1(a)) is applied to the column, washed with equilibrationbuffer, then eluted with a linear gradient to 500 mM sodium phosphate,pH 7.0 within 20 column volume. The run is repeated once under the sameconditions but with 3.75% PEG-6000, and a second time under the sameconditions but with 7.5% PEG-6000.

Example 5

Viral clearance via hydroxyapatite chromatography has been studied forat least 45 years [Shukla, Abhinav A. et al., Journal of Chromatography,B, 848:28-39 (2006); Pfefferkorn, E. R., and Hunter, H. S., Virology,20:433-445 (1963); Dove G B et al., Purification alternatives for IgM(human) monoclonal antibodies, p 194-209 in Ladisch M R, Builder S E,Painton, C C and Willson, R C (eds), ACS Symposium Series 427, AmericanChemical Society, Washington, D.C. (1998); Grun J B, White E M and SitoA F, Biopharm, 5:22-30 (1992)]. The mechanism of binding can be due toeither synergistic effects of multi-site binding [Aoyama, K. and Chiba,J., J. Imm. Meth., 162:201-210 (1993); Luellau, E. et al., Journal ofChromatography, A, 796:165-175 (1998); Josic D. et al., Biol. Chem.Hoppe Seylar, 372(3):149-156 (1991)], strong interactions of clusteredsurface phosphates (in the case of lipid enveloped viruses) with calcium[Sleigh, R W et al., J Dairy Res., 46:337-42 (1979); Gagnon, P.,BioProcess Int., 4(2):50-60 (2006)] or both. The original eluant usedalmost exclusively for hydroxyapatite was phosphate. However, sodiumchloride as an eluant has also been used for some 50 years [Hjerten, S.,Biochim. Biophys. Acta, 31:216-235 (1959)], with improvements noted forantibody purity [Giovannini, R. and Freitag, R., Biotechnol. Bioeng.,73:522-529 (2001); Schubert, S. and Freitag, R., J. Chromatogr., A.1142:106-113 (2007)] and aggregate removal [Gagnon, P., BioProcess Int.,4(2):50-60 (2006); Guerrier, L. et al., Journal of Chromatography, B:755:37-46 (2001)]. In more recent years, a variety of other additives,such as polyethylene glycol (PEG) [Gagnon, P. J. Immunol. Meth.,336:222-228 (2008)], have been employed as elution modifiers. PEG hasbeen shown to improve aggregate separation by differentially enhancingretention of larger solutes [Gagnon, P. J. Immunol. Meth., 336:222-228(2008)]. The size of MVM particles is approximately 18-26 nm [Crawford,L V. Virology, 29:605-612 (1966)] while that of X-MuLV is 80-110 nm.

A study performed several years ago [Ng, P. et al., “Monoclonal AntibodyPurification with CHT”, Genetic Engineering News, August 2006, Vol. 26,No. 14, p. 60; Gagnon, P. et al., Bio-Rad Laboratories Technical Note,2156 (2002); Gagnon, P. et al., “The practical task of purifyingantibodies with ceramic hydroxyapatite”, 11th Annual WatersideConference, May 1-3, Chicago (2006)] demonstrated >3 and 2 log clearanceof X-MuLV and MVM, respectively, in a sodium chloride gradient. Thecurrent study expands on these data and confirms that PEG providesadditional clearance X-MuLV and MVM at an antibody loading levelconsistent with current manufacturing processes.

Materials and Methods

Antibody and Viral Solutions

A monoclonal antibody, purified over a protein A column, was generouslysupplied by Avid Bioservices (Tustin, Calif., USA). The eluate wasneutralized to pH 7.0 and 0.5 M sodium phosphate, pH 7.0 was added to afinal concentration of 10 mM. Antibody was loaded onto CHT columns at˜10 mg protein/mL bed volume.

Stock solutions of either minute virus of mice (MVM) or xenotropicmurine leukemia virus (x-MuLV) (Charles River Laboratories, Malvern,Pa., USA) were used for this study.

CHT:

Ceramic hydroxyapatite (CHT™), type I, 40 micron, was supplied byBio-Rad Laboratories (Hercules, Calif., USA). The CHT was packed in11.8×100 mm columns (Atoll GmbH, Weingarten, Germany). For all steps,the linear flow rate was 300 cm/hr. All runs were performed at roomtemperature.

Chromatography Buffers:

The following buffers were employed for this study. Each buffer wastested for cytotoxicity and interference in the infectivity assaysystems used to quantitate each virus.

-   A) 10 mM sodium phosphate, pH 7.0-   B) 10 mM sodium phosphate, 10% PEG-1000 (Sigma-Aldrich, St. Louis,    Mo., USA), pH 7.0-   C) 10 mM sodium phosphate, 2 M NaCl, pH 7.0-   D) 30 mM sodium phosphate, 2 M NaCl, 10% PEG-1000, pH 7.0-   E) 600 mM potassium phosphate, pH 7.0    Running Conditions:    I. NaCl Elution

The column was equilibrated with Buffer A. Following sample applicationthe column was washed with 10 column volumes (CV) of Buffer A and elutedwith a 10 column volume gradient to Buffer C. The gradient was held at100% Buffer C until the antibody peak returned to baseline. The columnwas then cleaned with Buffer E and sanitized with 1 M NaOH. Eachexperiment was run in duplicate.

II. PEG/NaCl Elution

The CHT column was equilibrated with buffer A.

Equilibration and sample application were as described in the NaClelution above. The column was then washed with 10 CV of Buffer A and 5CV of Buffer B. Elution was a 10 column volume gradient to Buffer D. Thegradient was held at 100% Buffer D until the antibody peak returned tobaseline. The column was then cleaned with Buffer E and sanitized with 1M NaOH. Each experiment was run in duplicate.

Viral Testing

Aliquots of antibody were mixed in a 1:20 ratio with virus stocksolutions. A process control sample was removed and divided into twoaliquots (spiked load and hold control). The spiked load was testedimmediately; the hold control was kept at room temperature throughoutthe chromatography and then tested. Virus was also diluted into a mediacontrol and either assayed immediately or at the end of thechromatography (media control immediate and end samples, respectively).The rest of the aliquot was divided into two parts and chromatographedin duplicate. The flow-through+wash, eluate and column strip fractionswere collected during each run and tested immediately via infectivityassay. The amount of virus present in each sample was determined bydilution techniques and the log clearance for each sample calculatedfrom these data.

In some cases, samples were frozen and subsequently assayed for viralDNA via quantitative polymerase chain reaction. Each sample or controlwas amplified in triplicate for each dilution. Controls includedcellular RNA and water for preparation of viral CDNA and negativecontrol DNA and XuLV cDNA for amplification controls, as well as systemsuitability controls using β-actin. Amplification was performed using anApplied Biosystems Prism 7700 Sequence Detection System (LifeTechnologies, Carlsbad, Calif., USA). The number of viral equivalentsmeasured from these assays were then determined and log clearance valuesderived from these data.

Results and Conclusions

The infectivity assay results of the X-MuLV and MVM clearanceexperiments are shown in Tables 1 and 2, respectively. Quantitative PCRdata are shown in Table 3 and a summary of all data is presented inTable 4.

TABLE 1 Clearance of X-MuLV on CHT NaCl PEG/NaCl Total Virus ClearanceTotal Virus Clearance Sample (log₁₀) (Log₁₀) (log₁₀) (Log₁₀) MediaControl 7.28 — 6.93 — Immediate Spiked Load Run 1 7.07 — 7.19 —Flowthrough Run 1 3.57 3.50 3.94 3.25 Eluate Run 1 3.89 3.18 <3.15 >4.04Column Strip Run 1 6.20 0.87 6.16 1.03 Hold Control Run 1 7.27 −0.207.25 −0.06 Spiked Load Run 2 7.06 — 7.19 — Flowthrough Run 2 3.67 3.393.89 3.30 Eluate Run 2 3.39 3.67 <3.08 >4.11 Column Strip Run 2 6.310.75 5.87 1.32 Hold Control Run 2 7.27 −0.21 7.25 −0.06 Media ControlEnd 7.11 0.17 7.00 −0.07

TABLE 2 Clearance of MVM on CHT NaCl PEG/NaCl Total Virus ClearanceTotal Virus Clearance Sample (log₁₀) (Log₁₀) (log₁₀) (Log₁₀) MediaControl 8.95 — 8.81 — Immediate Spiked Load Run 1 9.28 — 9.15 —Flowthrough Run 1 8.55 0.73 8.69 0.46 Eluate Run 1 7.49 1.79 6.60 2.55Column Strip Run 1 8.83 0.45 8.67 0.48 Hold Control Run 1 9.16 0.12 9.080.07 Spiked Load Run 2 9.28 — 9.15 — Flowthrough Run 2 8.57 0.71 8.360.79 Eluate Run 2 7.68 1.60 6.39 2.76 Column Strip Run 2 8.87 0.41 8.760.39 Hold Control Run 2 9.16 0.12 9.08 0.07 Media Control End 8.90 0.058.70 0.11

TABLE 3 Quantitative PCR on X-MuLV clearance using NaCl + PEG SampleTotal Virus (Log₁₀) Clearance (Log₁₀) Spiked Load 10.67 — Hold Control10.72 −0.05 Eluate Run 1 6.66 4.01 Eluate Run 2 6.53 4.15

TABLE 4 Summary of viral clearance data (load to eluate) X-MuLV MVMClearance (Log₁₀) Clearance (Log₁₀) Condition Run 1 Run 2 Average Run 1Run 2 Average NaCl 3.18 3.67 3.43 1.79 1.60 1.70PEG/NaCl >4.04 >4.11 >4.08 2.55 2.76 2.66 PEG/NaCl 4.01 4.15 4.08 N/AN/A (qPCR) N/A, not assayed

The tight binding of viruses to CHT can be explained at least in part bythe multi-modal nature of hydroxyapatite interactions. For envelopedviruses, the significant number of phosphate groups on the lipidenvelope interact strongly with the calcium atoms via a chelationmechanism as has been previously shown for both proteins and nucleicacids [Sleigh, R W et al., J Dairy Res., 46:337-42 (1979); Gagnon, P.,BioProcess Int., 4(2):50-60 (2006)]. In general, the much larger surfacearea provided by viruses would be expected to result in significantlytighter binding as compared to much smaller bio-molecules (such asantibodies). Such an effect has been noted for IgG antibody aggregates,which generally elute later on hydroxyapatite in a gradient thanmonomeric IgG, and for larger antibody types such as IgA and IgM[Aoyama, K. and Chiba, J., J Imm. Meth., 162:201-210 (1993); Luellau, E.et al., Journal of Chromatography, A, 796:165-175 (1998); Josic D. etal., Biol. Chem. Hoppe Seylar, 372(3):149-156 (1991), Gagnon, P. J.Immunol. Meth., 336:222-228 (2008)]. The effect is similar to thatobserved on cation exchange resins (see, for example, 19).

The data from Tables 1 and 2 indicate, first, that the experimentalconditions in and of themselves did not inactivate either virus, norwere the viruses themselves inherently unstable since all hold controlshad the same virus titers to within 0.5 log as the initial measurements.In addition, approximately 80% or greater of the applied viruses in theloads were recovered in the fractions analyzed, indicating good massbalance for these studies. It should be noted that the majority of bothviruses appear in the column strip fractions.

The X-MuLV clearance in NaCl alone confirms the previously-reportedvalue of >3 logs [Gagnon, P. et al., “The practical task of purifyingantibodies with ceramic hydroxyapatite”, 11th Annual WatersideConference, May 1-3, Chicago (2006)] for this type of gradient elution.Clearance was increased by 0.6 log when PEG at a fixed concentration wasadded to the NaCl gradient. Similar confirmation of existing data withMVM clearance [Gagnon, P. et al., “The practical task of purifyingantibodies with ceramic hydroxyapatite”, 11th Annual WatersideConference, May 1-3, Chicago (2006)] in NaCl gradients was alsoobtained. Here, the use of PEG in combination with NaCl removed greaterthan 10 times more virus. This capability enables potentiallysignificant improvements in product safety.

The effect of PEG may reflect its previously-documented ability to causetighter binding of aggregates compared to monomers to a variety ofchromatographic supports [Luellau, E. et al., Journal of Chromatography,A, 796:165-175 (1998); Gagnon, P. et al., Chromatogr., A 743:51-55(1996)]. Because the number of plaques for the PEG/NaCl eluates fellbelow the quantitation level for X-MuLV, only minimum clearance valuescould be reported. To address this, quantitative PCR was performed onthese samples; the data reveal that just over 4 logs clearance wasobtained, in agreement with the plaque assay system.

These data indicate that CHT reliably removes X-MuLV up to 4 logs, andcan remove MVM in the range of 1-2 logs. The vast majority of therecovered X-MuLV is recovered in the regeneration fraction, which isobtained by stripping the column with a benign (neutral phosphate)buffer.

It will be understood by the person of ordinary skill in the art how toscale up the results from experiments such as those described in theabove examples, to whatever volume required to meet their particularrequirements. It will also be understood by such persons that otherapproaches to method development, such as high-throughput roboticapproaches, can be applied to determine the conditions that mosteffectively embody the invention for a particular antibody.

All references cited herein are incorporated by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupercede and/or take precedence over any such contradictory material.

All numbers expressing quantities of ingredients, chromatographyconditions, and so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired performance sought to beobtained by the present invention.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

1. A method for enhancing protein binding capacity of a mixed modechromatography support, the method comprising contacting said supportwith a protein preparation in the presence of an aqueous-solublenonionic organic polymer; and eluting a target protein from the supportin the presence of the aqueous-soluble nonionic organic polymer, wherein(a) the preparation comprises antibodies, and a non-antibody targetprotein that is substantially free from antibodies is collected and thenon-antibody target protein is submitted to at least one furtherpurification step; or (b) the preparation does not comprise antibodies.2. The method of claim 1, wherein the preparation comprises antibodiesand a non-antibody target protein that is substantially free fromantibodies is collected and the target protein is submitted to at leastone further purification step.
 3. The method of claim 1, wherein thepreparation does not comprise antibodies.
 4. The method of claim 1,wherein the nonionic organic polymer is from the group consisting ofdextran, starch, cellulose, polyvinylpyrrolidone, polypropylene glycoland polyethylene glycol (PEG).
 5. The method of claim 1, wherein thenonionic organic polymer comprises two or more nonionic inorganicpolymers.
 6. The method of claim 1, wherein the nonionic organic polymerhas an average molecular weight of 100 to 10,000 daltons.
 7. The methodof claim 1, wherein the mixed mode support exploits a combination of twoor more of the following functionalities to adsorb components of thepreparation: cation exchange, anion exchange, hydrophobic interaction,hydrophilic interaction, hydrogen bonding, pi-pi bonding, metalaffinity.
 8. The method of claim 1, wherein the mixed mode supportcomprises hydroxyapatite.
 9. The method of claim 8, wherein the mixedmode support is selected from the group consisting of hydroxyapatite CHTType I, 20 micron; hydroxyapatite CHT Type I, 40 micron; hydroxyapatiteCHT Type I, 80 micron; hydroxyapatite CHT Type II, 20 micron;hydroxyapatite CHT Type II, 40 micron; and hydroxyapatite CHT Type II,80 micron.
 10. The method of claim 1, wherein the mixed mode supportcomprises fluoroapatite.
 11. The method of claim 10, wherein the mixedmode support comprises fluoroapatite CFT Type I, 40 micron orfluoroapatite CFT Type II, 40 micron.
 12. The method of claim 1, whereinthe mixed mode support comprises a ligand selected from the groupconsisting of Capto-MMC, Capto-Adhere, Capto-S, Capto-Q, MEP Hypercel,and ABx.
 13. The method of claim 1, wherein the method comprises atleast one other purification step.
 14. The method of claim 1, whereinseparation of virus from the target protein on the mixed modechromatography support operated in bind elute mode is enhanced comparedto the separation that would occur in the absence of the aqueous-solublenonionic organic polymer.
 15. The method of claim 1, wherein retentionon the support of aggregates, virus, and other contaminants larger thanthe target protein is enhanced to a greater degree than retention of thetarget protein, and wherein separation of the aggregates, virus, andother contaminants from the target protein is thereby increased.
 16. Themethod of claim 1, wherein retention on the support of aggregates,virus, and other contaminants larger than the target protein is enhancedto a greater degree than retention of the target protein and wherein themethod allows for increased capacity for contaminant removal from thetarget protein.
 17. The method of claim 1, wherein retention on thesupport of the target protein is enhanced to a greater degree thanretention of molecules smaller than the target protein, and whereinseparation of the molecules from the target protein is therebyincreased.